PROJECT SUMMARY/ABSTRACT During the past decades, studies of heart development have been mainly focused on conserved gene regulatory mechanisms, which control various aspects of cardiogenesis across multiple species from drosophila to mouse. These conserved gene programs include cardiac transcriptional factors (such as Nkx2.5, Isl1, Tbx20), microRNAs, and epigenetic regulators. Despite the high evolutionary conservation of cardiogenesis, the human heart exhibits unique properties, including distinctive morphogenesis and electrophysiological properties. These species-specific differences suggest the existence of novel genetic programs in each species. As the limitation of experimental setting, it remains unclear the mechanisms that regulate unique aspects of human cardiogenesis, and how human-specific mechanisms interact with conserved gene programs to fine-tune human heart development. Recently, accumulating evidence demonstrated that long noncoding RNAs (lncRNAs) play important roles in cell fate specification and organogenesis, including the heart. LncRNAs are greater than 200bp non-coding transcripts, which account for >40% of human transcriptome. Many lncRNAs are tissue-specific and species-specific. Recently, we identified a novel human-specific lncRNA, named Heart Brake LncRNA1 (HBL1) (Dev. Cell. 2017, 4:333-8). HBL1 is highly expressed in both cytoplasm and nucleus of human induced pluripotent stem cells (hiPSCs). Cytosolic HBL1 modulates cardiomyocyte (CM) development from hiPSCs by counteracting microRNA1 (MIR1). Pluripotency marker gene SOX2 activates HBL1 transcription. In this proposal, we will analyze further, a novel mechanism of nuclear HBL1 in initiating the cardiac gene-expressing program via interacting with polycomb repressive complex 2 (PRC2). Additionally, we will test a hypothesis that HBL1 adds a new layer of human- specific regulatory mechanism on top of a conserved cardiogenic axis. All together, the central hypothesis is that transcriptional factor SOX2, lncRNA HBL1, microRNA-1 and PRC2 complex composite a whole network to control human cardiogenesis from pluripotent stem cells.